Protocol for Fixing Coral Tissues with PAXgene Tissue Fixative followed by EDTA Decalcification

The goal of this protocol is to fix tissue for downstream applications such as cryosectioning to create tissue sections that preserve structural integrity and maintain the integrity of RNA and proteins for downstream imaging and RNA extraction

Therefore, all of these steps should be performed with gloves and in a clean workspace. And, since we are working with fixative, all steps utilizing fixative and the several wash steps thereafter should be performed in the fume hood.

Supplies

1. PAXgene Tissue Fix Containers
2. PAXgene Tissue Stabilizer
   • Add ethanol to according to manufacturer’s instructions
3. Forceps
4. Small clippers
5. 0.5 M EDTA
6. Optional: Tissue Casettes

Preparation: Needs to happen daily for each step

1. Clean fume hood bench with clean paper towels (spray solution, wipe down) in the following order:
   • 10% bleach solution
   • DI water
   • 70% ethanol
   • RNAse cleaner (spray bottle)
2. Sterilize forceps and clippers in same way

Fixation

1. Fix branchlets in PAXgene fixative for 24 hours.

   1. Get coral from CBLS wetlab and gently transport upstairs in water from the aquarium

   2. Use sterilized clippers (see “Preparation” above) to snip off tissue of interest and use sterilzied forceps to transfer the tissue to a tissue casette (we may want to autoclave or RNAse treat these) and submerge in the PAXgene fixative container.

   3. Up to 4-6 small branches can fit in a tissue casette and each container can hold 4 casettes.

   4. Close the container and keep at room temp in the hood, for 24 hours.
      • You will see the pigments quickly leech out from the tissue

      

      

2. Transfer branchlets to PAXgene stabilizer at 4 ºC for 24 hours
   • Pour off PAXgene fixative into labelled waste container (in hood) and refill the container with PAXgene stabilizer. I usually pour off this first amount of stabilizer as a “wash” and then refill the container with PAXgene stabilizer again.
   • You may need to use forceps to make sure the tissue casettes or loose tissue do not fall out.
   • Put container in flammable fridge (4 ºC)
3. After 24 hours, set up the following beakers:
   1. 1 beaker with 1X PBS
      • if downstream applications are sensitive to RNAses, use RNAse free H2O and RNAse cleaned materials for all solutions and containers/materials
   2. 1 beaker with 0.5 M EDTA for decalcification
4. Wash tissue casette by submerging in the PBS beaker (use sterilized forceps to transfer)
   • Goal: remove excess PAXgene tissue stabilizer (contains ethanol), which forms a precipiate with the EDTA
   • Can open up tissue casettes to take image to track decalcification process, and optionally can perform additional washes
5. Transfer washed casette to beaker containing EDTA. Once all casettes are processed, cover beaker with parafilm (poke holes in case gas builds up) and place on a shaker at low speed (i.e. 150 rm) in the cold room (4 ºC) until decalcification is complete (for small branches this takes ~48 hours).
   • Change EDTA solution every 24 or 48 hours

   

   

   

   • Example of branches partially decalcified (24 hours)

   

   • Example of branches fully decalcified (48 hours)

   

6. When the samples are fully decalcified (no CaCO3 left visually, samples may float), rinse tissue in RNAse/DNAse free PBS
   • on 9/4/23 did a short rinse but a precipiate still formed when I put the tissue in the stabilizer, so a longer rinse, for example 10 minutes at 4 ºC, is recommended
   • alternatievly: wash in PBS, then transfer to tube with stabilizer, then after 5 minutes remove stabilier and any precipiate that formed and replace with fresh stabilizer
7. Transfer branchlets to PAXgene stabilizer for 24 hours
   • set up 2mL screw-cap tubes with 1 mL of stabilizer, add one branch per tube
   • either incubate at RT or 4ºC for 24 hours to allow for stabilizer to penetrate and then transfer to -80 ºC, or put straight in -80 ºC, need to figure out what is better